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Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits (Oryctolagus cuniculus)?
ARTICLE IN PRESS
www.elsevier.de/mambio
Original investigation
Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits (Oryctolagus cuniculus)? By Adela Mendoza, Valeria Guzma´n, Carolina Rojas and Robyn Hudson Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Mexico City, Mexico Receipt of Ms. 16.1.2004 Acceptance of Ms. 7.6.2004
Abstract Rabbit pups are only nursed for about 3 min once a day. They depend on a pheromone on the mother’s ventrum to locate nipples and on tactile stimulation of the muzzle to grasp them. In a continuing study of the sensory input guiding suckling behavior we investigated the whisker array in newborn pups and the possible contribution of the whiskers to suckling. Rabbits are born with approximately 76 whiskers arranged in seven to nine rows and increasing in length from rostral to caudal. No significant difference was found between pups with whiskers cut and intact controls in latency to perform the stereotyped nipple-search behavior, latency to attach to nipples, time spent on nipples, milk ingested, or in the strength of conditioning to a novel odor paired with suckling. Thus, the whiskers do not seem important for suckling in newborn rabbits. r 2005 Elsevier GmbH. All rights reserved. Key words: Oryctolagus cuniculus, suckling, vibrissae, altricial mammals
Introduction For newborn mammals the task of finding, attaching to and successfully sucking a nipple or teat is one of the principle challenges of postnatal life. The European rabbit (Oryctolagus cuniculus) is a notable example. Immediately after giving birth, female rabbits leave their altricial young and only return to nurse them for about 3–4 min every 24 h (reviewed in Hudson and Distel 1982, 1989). The young, in strong competition with littermates (Drummond et al. 2000), drink up to 25% of their body weight in this short time (Lincoln 1974; Hudson et al. 1996). A pheromone on the mother’s ventrum is essential for the release of the stereotyped nipple-search behavior and to guide pups to nipples (Hudson and Distel 1983, 1995). In addition, at the
nipples somatosensory input to the muzzle is then essential for nipple attachment (Distel and Hudson 1985). Perioral stimulation is also important for learning novel odors associated with suckling. If the mother’s ventrum is scented with an artificial odorant, pups learn to associate the novel odor with suckling in just one 3-min nursing episode. When tested 24 h later on an animal or a fur scented with the odorant, conditioned but not control pups show the stereotyped nipple-search response (Hudson 1985; Kindermann et al. 1991, 1994; Allingham et al. 1999). Not milk but rather perioral stimulation from sucking is the main reinforcer of such learning (Hudson et al. 2002).
1616-5047/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2004.06.005 Mamm. biol. 70 (2005) 2 110–116
ARTICLE IN PRESS Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits
In the present study, we asked whether in rabbits the whiskers play a role in sucklingassociated behaviors since the pups are born with a conspicuous array of these. In contrast to adult mammals (Brecht et al. 1997; Landers and Sullivan 1999a; Cardenas et al. 2001; Krupa et al. 2001), there have been few behavioral studies of whisker function in newborns (but see Landers and Sullivan 1999a, b), and to our knowledge, none in a natural biological context. It was therefore our aim (1) to provide a description of the whisker array in the newborn rabbit, (2) to investigate the effect of removing the whiskers on nipple-search behavior and the learning of olfactory cues associated with suckling, and (3) to investigate the effect of removing the whiskers on suckling performance under natural conditions in the nest.
Material and methods The following three experimental sets were performed:
The whiskers Four two-day-old chinchilla-breed pups (day 0=day of birth) from four litters, weighing 69.4–96 g, and killed with an i.p. overdose of pentobarbital (Sedal-Vets, Ttokkyo Laboratories) were used. Under a magnifying lamp each whisker was plucked and using fine calipers and a Vernier scale, its length was recorded and entered onto the whisker map for that particular pup. In five cases whiskers broke during plucking and were excluded from the analysis. To estimate the length of the subcutaneous portion, in two additional pups the whiskers were dyed, plucked, and the undyed section measured. Depilatory cream was then applied for 3 min and the skin wiped clean and rubbed with graphite to enhance visibility of the vibrissal pores. Under the magnifying lamp the pores on both sides of the muzzle were mapped onto a schematic representation of the mystacial area.
Nipple-search behavior and olfactory learning Thirty-two two-day-old chinchilla-breed pups from 11 litters were used. On day 1 they were separated from the mother to ensure high motivation in the
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nipple-search task the following day, and colormarked in the ears for individual identification. At 10:00 h on the morning of day 2 they were weighed and assigned to one of two groups (n=16/group) in such a way as to balance weights between treatments; experimental pups whose whiskers were cut 1 h before testing (group W; mean weight 62.8 g, SE 3.0) and control pups with whiskers intact (group W+; mean weight 60.4 g, SE 2.4; two-tailed t-test for difference in weight between groups: t ¼ 0:65; df 30, P40.62). Whiskers were cut rather than plucked, cauterized or removed with depilatory cream to avoid damage to the fine mystacial skin. Removing whiskers by cutting is sufficient to disrupt a variety of whisker-mediated behaviors in adult rats (Brecht et al. 1997; Cardenas et al. 2001; Krupa et al. 2001). At 11:00 h the pups were exposed individually to a standard olfactory conditioning procedure (Hudson et al. 2002). Experimental and control pups were placed alternately on the ventrum of an anesthetized lactating doe restrained on her back in a Plexiglas trough forming an arena enclosing the six rear nipples (Hudson 1985; Hudson et al. 2002). The doe’s ventral fur and area around the nipples had been lightly perfumed with the cologne Chanel No. 5 (Hudson 1985; Hudson and Distel 1986, 1987; Kindermann et al. 1991, 1994; Hudson et al. 2002). Pups were allowed to move freely and to attach to and suck 10 nipples for a maximum of 10 s each. Their behavior was recorded using a video camera mounted above the arena, and latencies to search, to attach to nipples and the duration of attachments were scored. The next day, pups were given a recall test in which they were placed individually for 5 min on a tanned rabbit fur warmed to 30 1C enclosed by an acrylic arena (13 18 height 13 cm3), and perfumed with three spots of Chanel placed 2 cm apart along the central long axis (Kindermann et al. 1991, 1994; Allingham et al. 1999; Hudson et al. 2002; Fig. 3). Latency to start the stereotyped nipple-search behavior, time taken to initiate search bouts, total search time, and the total time searching was performed within the stimulus area defined as an ellipse enclosing the Chanel spots (Hudson et al. 2002; Fig. 3) were scored.
Suckling performance Six chinchilla-breed litters standardized to 10 pups each were used. Ten pups represents the upper end of the range in litter size for rabbits and was chosen to ensure competition among pups during suckling (Drummond et al. 2000). On the day of birth (day 0) pups were separated from their mother, colormarked in the ears for individual identification,
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weighed, and assigned in alternating order of weight to one of two groups. In group W (mean birth weight 48.8 g, SE 1.3) pups’ whiskers were cut, while in group W+ (mean birth weight 49.3 g, SE 1.5) the whiskers were left intact. Difference in birth weight between the two groups was not significant (two-tailed t-test: t ¼ 0:51; df 52, P40.61). At 10:00 h each day until day 8, pups were stimulated to urinate by brushing the anogenital area with iced water, they were weighed, the whiskers again cut in group W, and at 11:00 h they were brought to the mother to be nursed. Immediately after nursing they were again weighed to determine milk intake. Experiments were conducted according to the Guidelines for Animal Care of the National Institutes of Health (USA, 1986) and the current laws of Mexico.
Results The whiskers The whiskers were arranged in seven to nine clearly distinguishable rows, which were not always symmetrical on the two sides of the muzzle (Figs. 1C and 2). The number of whiskers per side ranged from 33 to 40, resulting in a mean total of 75.8, SE 1.9 whiskers per pup. Whereas the shorter anterior whiskers formed a fan almost perpendicular to the muzzle the longer caudal ones sloped in a ventrocaudal direction closer to the face (Fig. 1A). In live pups, spontaneous movement of the whiskers or movement in response to gentle brushing or to mechanical deflection was never seen. While the subcutaneous portion of all whiskers was
about 1 mm, external whisker length varied from 2.5 to 18 mm (mean 7.8, SE 0.2), with a consistent increase in length from rostral to caudal (Fig. 2). Nipple-search behavior and olfactory learning When placed on the ventrum of the perfumed doe for odor conditioning, pups in both groups showed nipple-search behavior within a few seconds. There was no significant difference between groups in latencies to start searching across the 10 trials (mean 2.6 s, SE 1.4 vs. mean 3.0 s, SE 1.2 for W+ and W, respectively; one-tailed t-test: t ¼ 0:90; df 30, P40.19), or time taken to attach to nipples (mean 7.4 s, SE 0.6 vs. mean 7.9 s, SE 1.0; one-tailed t-test: t ¼ 0:47; df 30, P ¼ 0:32). When tested on the perfumed fur 24 h after conditioning, pups of both groups showed bouts of vigorous nipple-search behavior interrupted by quiescence or general locomotor activity. No significant difference was found between pups with whiskers (W+) and pups without whiskers (W) in latency to start or to resume searching (mean 26.0 s, SE 6.2 vs. mean 32.0 s, SE 11.0; one-tailed t-test: t ¼ 0:48; df 30, P40.32), in total time spent searching (mean 18.1 s, SE 3.7 vs. mean 15.6 s, SE 2.1; one-tailed t-test: t ¼ 0:56; df 30, P40.29), in time searching within the stimulus area (mean 10.9 s, SE 3.3 vs. mean 9.0 s, SE 1.7; one-tailed t-test: t ¼ 0:26; df 30, P40.40), or in the amount of time spent searching inside or outside the stimulus area
Fig. 1. Whisker array in a two-day-old rabbit. (A) Natural view. (B) The vibrissal pores after removing the whiskers with depilatory cream and rubbing the mystacial skin with graphite. (C) Drawing of the mystacial area of the same pup showing the arrangement of the whisker rows. Whisker length is indicated by the size of the dots using the same categories as in Fig. 2; the larger the dots, the longer the whiskers.
ARTICLE IN PRESS Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits
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77 g
96 g
x x
69 g
72 g
x
x x
2.5-4 mm 12-18 mm
4.5-5.5 mm
6-9.5 mm
10-11mm
x whisker broken during plucking
Fig. 2. Schematic representation of the whisker arrangement in four two-day-old pups from four litters. Pups’ weight at the time of measurement is given at the top left of each map. External whisker length is indicated by the size of the dots; the larger the dots, the longer the whiskers.
calculated as the per cent of time pups spent in these two areas (mean search time inside: 22%, SE 4.4 vs. mean 24%, SE 5.7; mean search time outside: 3%, SE 0.5 vs. mean 3%, SE 0.4; one-tailed t-test: t ¼ 0:55; df 30, P40.29; Fig. 3).
mean 164%, SE 12.6%; one-tailed t-test; t ¼ 0:26; df 52, P40.40), or in body weight at the end of the experiment (mean 92.4 g, SE 3.6 vs. mean 88.2, SE 4.0; one-tailed t-test: t ¼ 0:77; df 52, P40.23).
Suckling performance
Discussion
Nursing behavior appeared normal throughout the experiment. As soon as the box containing the pups was placed in the does’ cage, they entered it and remained nursing for no longer than 5 min. No significant difference was found between W+ and W pups in the daily amount of milk ingested (two-way ANOVA with repeated measures: ‘‘group’’ F ð1;52Þ ¼ 0:12; P ¼ 0:73; ‘‘days’’ F ð7;364Þ ¼ 12:17; Po0.0001; interaction ‘‘group’’ and ‘‘days’’ F ð7;364Þ ¼ 1:95; P ¼ 0:06), in the total amount of milk obtained by the end of the experiment (mean 82.6 g, SE 5.9 vs. mean 81.1 g, SE 6.1; one-tailed t-test: t ¼ 0:35; df 52, P40.37), including when this was normalized by expressing it as per cent of birth weight (mean 163%, SE 13.5% vs.
Although to our knowledge a description of the whiskers is lacking for other mammalian young, the general arrangement in newborn rabbits is consistent with the description for the adult rabbit (Swardlow 1991) and other species, supporting the view that the vibrissal apparatus is highly conserved across taxa (Brecht et al. 1997). However, the arrangement was more variable than in the adult rat or cat (Nomura et al. 1986; Brecht et al. 1997; Krupa et al. 2001), with the number of rows and of whiskers varying not only among individuals but also between the two sides of the face. It surprised us that cutting the whiskers had so little effect on locating nipples and suckling since in adult animals cutting
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30
25
Time (%)
20
15 Inside
10
Outside 5
0
W+
WTreatments
Fig. 3. Percent of time inside or outside the stimulus area in the recall test that was spent in nipple-search behavior by pups with whiskers (W+) and by pups without whiskers (W). The inset shows the ellipse (20% of the arena surface) enclosing the three spots of Chanel that was used to define the stimulus area. Means and SEs are given.
whiskers disrupts a variety of behaviors (Brecht et al. 1997; Cardenas et al. 2001; Krupa et al. 2001). The steps leading to successful suckling are among the most complex and precise orienting behaviors performed by newborn rabbits, requiring them to rapidly establish and maintain contact with the mother’s ventrum, to perform a particular pattern of head movements through her fur, to locate a nipple, to grasp and remain attached to it, and this repeatedly since the pups typically switch nipples several times within one nursing episode (Hudson and Distel 1983). As for the altricial young of other mammals (Hofer et al. 1981; Larson and Stein 1984; Blass et al. 1988; Morrow–Tesch and McGlone 1990) somatosensory input from the perioral area is critical for successful suckling. However, in rabbit pups this seems to come from the skin and lips rather than from the whiskers, and also seems to apply to the reinforcing role of perioral stimulation in learning to associate a novel odor with suckling (Hudson et al. 2002). Although the results are consistent with our failure to find evidence of movement of the
whiskers or of ‘‘whisking’’ in these young animals, they are surprising given the finding that rat pups rapidly learn to associate whisker stimulation with a novel reinforcer (Landers and Sullivan 1999a, b), and after cutting the whiskers on postnatal day 3 or 4, take longer to attach to nipples than intact controls (Young and Sullivan, congress abstract, cited in Landers and Sullivan 1999a). The lack of effect in rabbit pups may be due to their extreme dependence on odor cues on the mother’s ventrum for the release and guidance of the stereotyped nipple-search behavior (Hudson and Distel 1983, 1995; Distel and Hudson 1985).
Acknowledgements Support from PAPIIT Grant IN217100 is gratefully acknowledged and we thank Daniel Paul Alarcon Tinoco for photographic assistance in preparing Fig. 1, and Hans Distel for help with the German abstract.
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Zusammenfassung Tragen die Schnurrhaare bei neugeborenen Kaninchen (Oryctolgus cuniculus) zum Zitzensuchund Saugverhalten bei? Jungkaninchen werden nur einmal am Tag fu¨r etwa drei Minuten gesa¨ugt. Fu¨r das Auffinden der Zitzen sind sie auf ein Pheromon auf der Bauchhaut der Mutter angewiesen, und fu¨r deren Ergreifen auf periorale taktile Reize. In Fortsetzung unserer Untersuchungen zur sensorischen Kontrolle dieses Verhaltens beschreiben wir die Anordnung der Schnurrhaare bei neugeborenen Kaninchen und untersuchen ihren mo¨glichen Beitrag zum Sa¨ugeverhalten. Bei der Geburt besitzen Jungkaninchen etwa 76, in 7–9 Reihen angeordnete Schnurrhaare, deren La¨nge von rostral nach caudal zunimmt. Nach Abschneiden der Schnurrhaare wurden keine signifikanten Unterschiede zwischen Jungen mit und ohne Schnurrhaare beobachtet, sowohl in der Latenz des Zitzensuchens, des Zitzenfassens, wie in der Besaugungsdauer, der erhaltenen Milchmenge oder dem Grad der Konditionierung auf einen neuen Geruchsstoff. Demnach spielen bei neugeborenen Kaninchen die Schnurrhaare keine wesentliche Rolle fu¨r das Auffinden und Besaugen von Zitzen. r 2005 Elsevier GmbH. All rights reserved.
References Allingham, K.; Brennan, P. A.; Distel, H.; Hudson, R. (1999): Expression of c-Fos in the main olfactory bulb of neonatal rabbits in response to garlic as a novel and conditioned odour. Behav. Brain. Res. 104, 157–167. Blass, E. M.; Shuleikina-Turpaeva, K.; Luschekin, V. (1988): Sensory determinants of nippleattachment behavior in 2–4-day-old kittens. Dev. Psychobiol. 21, 365–370. Brecht, M.; Preilowski, B.; Mezenich, M. M. (1997): Functional architecture of the mystacial vibrissae. Behav. Brain. Res. 84, 81–97. Cardenas, F.; Lamprea, M. R.; Morato, S. (2001): Vibrissal sense is not the main sensory modality in rat exploratory behavior in the elevated plusmaze. Behav. Brain Res. 122, 169–174. Distel, H.; Hudson, R. (1985): The contribution of the olfactory and tactile modalities to the nipple-search behaviour of newborn rabbits. J. Comp. Physiol. A 157, 599–605. Drummond, H.; Va´zquez, E.; Sa´nchez-Colo´n, S.; Martı´ nez-Go´mez, M.; Hudson, R. (2000): Competition for milk in the domestic rabbit: survivors benefit from littermate deaths. Ethology 106, 511–526. Hofer, M. A.; Fischer, A.; Shair, H. (1981): Effects of infraorbital nerve section on survival, growth and suckling behaviors of developing rats. J. Comp. Physiol. Psychol. 95, 123–133. Hudson, R. (1985): Do newborn rabbits learn the odor stimuli releasing nipple-search behavior? Dev. Psychobiol. 18, 575–585. Hudson, R.; Distel, H. (1982): The pattern of behaviour of rabbit pups in the nest. Behaviour 79, 255–271.
Hudson, R.; Distel, H. (1983): Nipple location by newborn rabbits: behavioural evidence for pheromonal guidance. Behaviour 85, 260–275. Hudson, R.; Distel, H. (1986): Pheromonal release of suckling in rabbits does not depend on the vomeronasal organ. Physiol. Behav. 37, 123–128. Hudson, R.; Distel, H. (1987): Regional autonomy in the peripheral processing of odor signals in newborn rabbits. Brain. Res. 421, 85–94. Hudon, R.; Distel, H. (1989): Temporal pattern of suckling in rabbit pups: a model of circadian synchrony between mother and young. In: Development of Circadian Rhythmicity and Photoperiodism in Mammals, Ed. by S. M. Reppert. Boston: Perinatology Press, Pp. 83–102. Hudson, R.; Distel, H. (1995): On the nature and action of the rabbit nipple-search pheromone: a review. In: Chemical Signals in Vertebrates VII, Ed. by A. Apfelbach, D. Mu¨ller-Schwarze, K. Reuter, E. Weiler. Oxford: Pergamon Press, Pp. 223–232. Hudson, R.; Bilko´, A´.; Alltba¨cker, V. (1996): Nursing, weaning and the development of independent feeding in the rabbit (Oryctolagus cuniculus). Z. Sa¨ugetierkunde 61, 39–48. Hudson, R.; Labra-Cardero, D.; Mendoza-Soylovna, A. (2002): Sucking, not milk, is important for the rapid learning of nipple-search odors in newborn rabbits. Dev. Psychobiol. 41, 226–235. Kindermann, U.; Gervais, R.; Hudson, R. (1991): Rapid odor conditioning in newborn rabbits: amnesic effect of hypothermia. Physiol. Behav. 50, 457–460.
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Kindermann, U.; Hudson, R.; Distel, H. (1994): Learning of suckling odors by newborn rabbits declines with age and suckling experience. Dev. Psychobiol. 27, 111–122. Krupa, D. J.; Matell, M. S.; Brisben, A. J.; Oliveira, L. M.; Nicolelis, M. A. L. (2001): Behavioral properties of the trigeminal somatosensory system in rats performing whiskerdependent tactile discriminations. J. Neurosci. 21, 5752–5763. Landers, M. S.; Sullivan, R. M. (1999a): Vibrissaeevoked behavior and conditioning before functional ontogeny of the somatosensory vibrissae cortex. J. Neurosci. 19, 5131–5137. Landers, M. S.; Sullivan, R. M. (1999b): Norepinepherine and associative conditioning in the neonatal rat somatosensory system. Dev. Brain. Res. 114, 261–264. Larson, M. A.; Stein, B. E. (1984): The use of tactile and olfactory cues in neonatal orientation and localization of the nipple. Dev. Psychobiol. 17, 423–436. Lincoln, D. W. (1974): Suckling: a time-constant in the nursing behaviour of the rabbit. Physiol. Behav. 13, 711–714.
Morrow-Tesch, J.; McGlone, J. L. (1990): Sensory systems and nipple attachment behavior in neonatal pigs. Physiol. Behav. 47, 1–4. Nomura, S.; Itoh, K.; Sugimoto, T.; Yasui, Y.; Kamiya, H.; Mizuno, N. (1986): Mystacial vibrissae representation within the trigeminal sensory nuclei of the cat. J. Comp. Neurol. 253, 121–133. Swardlow, H. A. (1991): Efferent neurons and suspected interneurons in second somatosensory cortex of the awake rabbit: receptive fields and axonal properties. J. Neurophysiol. 66, 1392–1409.
Authors’ address: Adela Mendoza, Valeria Guzma´n, Carolina Rojas, and Robyn Hudson, Departamento de Biologı´ a Celular y Fisiologı´ a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Apartado Postal 70228, CP 04510 Me´xico, D.F., Mexico. (e-mail: [email protected].)
www.elsevier.de/mambio
Original investigation
Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits (Oryctolagus cuniculus)? By Adela Mendoza, Valeria Guzma´n, Carolina Rojas and Robyn Hudson Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Mexico City, Mexico Receipt of Ms. 16.1.2004 Acceptance of Ms. 7.6.2004
Abstract Rabbit pups are only nursed for about 3 min once a day. They depend on a pheromone on the mother’s ventrum to locate nipples and on tactile stimulation of the muzzle to grasp them. In a continuing study of the sensory input guiding suckling behavior we investigated the whisker array in newborn pups and the possible contribution of the whiskers to suckling. Rabbits are born with approximately 76 whiskers arranged in seven to nine rows and increasing in length from rostral to caudal. No significant difference was found between pups with whiskers cut and intact controls in latency to perform the stereotyped nipple-search behavior, latency to attach to nipples, time spent on nipples, milk ingested, or in the strength of conditioning to a novel odor paired with suckling. Thus, the whiskers do not seem important for suckling in newborn rabbits. r 2005 Elsevier GmbH. All rights reserved. Key words: Oryctolagus cuniculus, suckling, vibrissae, altricial mammals
Introduction For newborn mammals the task of finding, attaching to and successfully sucking a nipple or teat is one of the principle challenges of postnatal life. The European rabbit (Oryctolagus cuniculus) is a notable example. Immediately after giving birth, female rabbits leave their altricial young and only return to nurse them for about 3–4 min every 24 h (reviewed in Hudson and Distel 1982, 1989). The young, in strong competition with littermates (Drummond et al. 2000), drink up to 25% of their body weight in this short time (Lincoln 1974; Hudson et al. 1996). A pheromone on the mother’s ventrum is essential for the release of the stereotyped nipple-search behavior and to guide pups to nipples (Hudson and Distel 1983, 1995). In addition, at the
nipples somatosensory input to the muzzle is then essential for nipple attachment (Distel and Hudson 1985). Perioral stimulation is also important for learning novel odors associated with suckling. If the mother’s ventrum is scented with an artificial odorant, pups learn to associate the novel odor with suckling in just one 3-min nursing episode. When tested 24 h later on an animal or a fur scented with the odorant, conditioned but not control pups show the stereotyped nipple-search response (Hudson 1985; Kindermann et al. 1991, 1994; Allingham et al. 1999). Not milk but rather perioral stimulation from sucking is the main reinforcer of such learning (Hudson et al. 2002).
1616-5047/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2004.06.005 Mamm. biol. 70 (2005) 2 110–116
ARTICLE IN PRESS Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits
In the present study, we asked whether in rabbits the whiskers play a role in sucklingassociated behaviors since the pups are born with a conspicuous array of these. In contrast to adult mammals (Brecht et al. 1997; Landers and Sullivan 1999a; Cardenas et al. 2001; Krupa et al. 2001), there have been few behavioral studies of whisker function in newborns (but see Landers and Sullivan 1999a, b), and to our knowledge, none in a natural biological context. It was therefore our aim (1) to provide a description of the whisker array in the newborn rabbit, (2) to investigate the effect of removing the whiskers on nipple-search behavior and the learning of olfactory cues associated with suckling, and (3) to investigate the effect of removing the whiskers on suckling performance under natural conditions in the nest.
Material and methods The following three experimental sets were performed:
The whiskers Four two-day-old chinchilla-breed pups (day 0=day of birth) from four litters, weighing 69.4–96 g, and killed with an i.p. overdose of pentobarbital (Sedal-Vets, Ttokkyo Laboratories) were used. Under a magnifying lamp each whisker was plucked and using fine calipers and a Vernier scale, its length was recorded and entered onto the whisker map for that particular pup. In five cases whiskers broke during plucking and were excluded from the analysis. To estimate the length of the subcutaneous portion, in two additional pups the whiskers were dyed, plucked, and the undyed section measured. Depilatory cream was then applied for 3 min and the skin wiped clean and rubbed with graphite to enhance visibility of the vibrissal pores. Under the magnifying lamp the pores on both sides of the muzzle were mapped onto a schematic representation of the mystacial area.
Nipple-search behavior and olfactory learning Thirty-two two-day-old chinchilla-breed pups from 11 litters were used. On day 1 they were separated from the mother to ensure high motivation in the
111
nipple-search task the following day, and colormarked in the ears for individual identification. At 10:00 h on the morning of day 2 they were weighed and assigned to one of two groups (n=16/group) in such a way as to balance weights between treatments; experimental pups whose whiskers were cut 1 h before testing (group W; mean weight 62.8 g, SE 3.0) and control pups with whiskers intact (group W+; mean weight 60.4 g, SE 2.4; two-tailed t-test for difference in weight between groups: t ¼ 0:65; df 30, P40.62). Whiskers were cut rather than plucked, cauterized or removed with depilatory cream to avoid damage to the fine mystacial skin. Removing whiskers by cutting is sufficient to disrupt a variety of whisker-mediated behaviors in adult rats (Brecht et al. 1997; Cardenas et al. 2001; Krupa et al. 2001). At 11:00 h the pups were exposed individually to a standard olfactory conditioning procedure (Hudson et al. 2002). Experimental and control pups were placed alternately on the ventrum of an anesthetized lactating doe restrained on her back in a Plexiglas trough forming an arena enclosing the six rear nipples (Hudson 1985; Hudson et al. 2002). The doe’s ventral fur and area around the nipples had been lightly perfumed with the cologne Chanel No. 5 (Hudson 1985; Hudson and Distel 1986, 1987; Kindermann et al. 1991, 1994; Hudson et al. 2002). Pups were allowed to move freely and to attach to and suck 10 nipples for a maximum of 10 s each. Their behavior was recorded using a video camera mounted above the arena, and latencies to search, to attach to nipples and the duration of attachments were scored. The next day, pups were given a recall test in which they were placed individually for 5 min on a tanned rabbit fur warmed to 30 1C enclosed by an acrylic arena (13 18 height 13 cm3), and perfumed with three spots of Chanel placed 2 cm apart along the central long axis (Kindermann et al. 1991, 1994; Allingham et al. 1999; Hudson et al. 2002; Fig. 3). Latency to start the stereotyped nipple-search behavior, time taken to initiate search bouts, total search time, and the total time searching was performed within the stimulus area defined as an ellipse enclosing the Chanel spots (Hudson et al. 2002; Fig. 3) were scored.
Suckling performance Six chinchilla-breed litters standardized to 10 pups each were used. Ten pups represents the upper end of the range in litter size for rabbits and was chosen to ensure competition among pups during suckling (Drummond et al. 2000). On the day of birth (day 0) pups were separated from their mother, colormarked in the ears for individual identification,
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weighed, and assigned in alternating order of weight to one of two groups. In group W (mean birth weight 48.8 g, SE 1.3) pups’ whiskers were cut, while in group W+ (mean birth weight 49.3 g, SE 1.5) the whiskers were left intact. Difference in birth weight between the two groups was not significant (two-tailed t-test: t ¼ 0:51; df 52, P40.61). At 10:00 h each day until day 8, pups were stimulated to urinate by brushing the anogenital area with iced water, they were weighed, the whiskers again cut in group W, and at 11:00 h they were brought to the mother to be nursed. Immediately after nursing they were again weighed to determine milk intake. Experiments were conducted according to the Guidelines for Animal Care of the National Institutes of Health (USA, 1986) and the current laws of Mexico.
Results The whiskers The whiskers were arranged in seven to nine clearly distinguishable rows, which were not always symmetrical on the two sides of the muzzle (Figs. 1C and 2). The number of whiskers per side ranged from 33 to 40, resulting in a mean total of 75.8, SE 1.9 whiskers per pup. Whereas the shorter anterior whiskers formed a fan almost perpendicular to the muzzle the longer caudal ones sloped in a ventrocaudal direction closer to the face (Fig. 1A). In live pups, spontaneous movement of the whiskers or movement in response to gentle brushing or to mechanical deflection was never seen. While the subcutaneous portion of all whiskers was
about 1 mm, external whisker length varied from 2.5 to 18 mm (mean 7.8, SE 0.2), with a consistent increase in length from rostral to caudal (Fig. 2). Nipple-search behavior and olfactory learning When placed on the ventrum of the perfumed doe for odor conditioning, pups in both groups showed nipple-search behavior within a few seconds. There was no significant difference between groups in latencies to start searching across the 10 trials (mean 2.6 s, SE 1.4 vs. mean 3.0 s, SE 1.2 for W+ and W, respectively; one-tailed t-test: t ¼ 0:90; df 30, P40.19), or time taken to attach to nipples (mean 7.4 s, SE 0.6 vs. mean 7.9 s, SE 1.0; one-tailed t-test: t ¼ 0:47; df 30, P ¼ 0:32). When tested on the perfumed fur 24 h after conditioning, pups of both groups showed bouts of vigorous nipple-search behavior interrupted by quiescence or general locomotor activity. No significant difference was found between pups with whiskers (W+) and pups without whiskers (W) in latency to start or to resume searching (mean 26.0 s, SE 6.2 vs. mean 32.0 s, SE 11.0; one-tailed t-test: t ¼ 0:48; df 30, P40.32), in total time spent searching (mean 18.1 s, SE 3.7 vs. mean 15.6 s, SE 2.1; one-tailed t-test: t ¼ 0:56; df 30, P40.29), in time searching within the stimulus area (mean 10.9 s, SE 3.3 vs. mean 9.0 s, SE 1.7; one-tailed t-test: t ¼ 0:26; df 30, P40.40), or in the amount of time spent searching inside or outside the stimulus area
Fig. 1. Whisker array in a two-day-old rabbit. (A) Natural view. (B) The vibrissal pores after removing the whiskers with depilatory cream and rubbing the mystacial skin with graphite. (C) Drawing of the mystacial area of the same pup showing the arrangement of the whisker rows. Whisker length is indicated by the size of the dots using the same categories as in Fig. 2; the larger the dots, the longer the whiskers.
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77 g
96 g
x x
69 g
72 g
x
x x
2.5-4 mm 12-18 mm
4.5-5.5 mm
6-9.5 mm
10-11mm
x whisker broken during plucking
Fig. 2. Schematic representation of the whisker arrangement in four two-day-old pups from four litters. Pups’ weight at the time of measurement is given at the top left of each map. External whisker length is indicated by the size of the dots; the larger the dots, the longer the whiskers.
calculated as the per cent of time pups spent in these two areas (mean search time inside: 22%, SE 4.4 vs. mean 24%, SE 5.7; mean search time outside: 3%, SE 0.5 vs. mean 3%, SE 0.4; one-tailed t-test: t ¼ 0:55; df 30, P40.29; Fig. 3).
mean 164%, SE 12.6%; one-tailed t-test; t ¼ 0:26; df 52, P40.40), or in body weight at the end of the experiment (mean 92.4 g, SE 3.6 vs. mean 88.2, SE 4.0; one-tailed t-test: t ¼ 0:77; df 52, P40.23).
Suckling performance
Discussion
Nursing behavior appeared normal throughout the experiment. As soon as the box containing the pups was placed in the does’ cage, they entered it and remained nursing for no longer than 5 min. No significant difference was found between W+ and W pups in the daily amount of milk ingested (two-way ANOVA with repeated measures: ‘‘group’’ F ð1;52Þ ¼ 0:12; P ¼ 0:73; ‘‘days’’ F ð7;364Þ ¼ 12:17; Po0.0001; interaction ‘‘group’’ and ‘‘days’’ F ð7;364Þ ¼ 1:95; P ¼ 0:06), in the total amount of milk obtained by the end of the experiment (mean 82.6 g, SE 5.9 vs. mean 81.1 g, SE 6.1; one-tailed t-test: t ¼ 0:35; df 52, P40.37), including when this was normalized by expressing it as per cent of birth weight (mean 163%, SE 13.5% vs.
Although to our knowledge a description of the whiskers is lacking for other mammalian young, the general arrangement in newborn rabbits is consistent with the description for the adult rabbit (Swardlow 1991) and other species, supporting the view that the vibrissal apparatus is highly conserved across taxa (Brecht et al. 1997). However, the arrangement was more variable than in the adult rat or cat (Nomura et al. 1986; Brecht et al. 1997; Krupa et al. 2001), with the number of rows and of whiskers varying not only among individuals but also between the two sides of the face. It surprised us that cutting the whiskers had so little effect on locating nipples and suckling since in adult animals cutting
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30
25
Time (%)
20
15 Inside
10
Outside 5
0
W+
WTreatments
Fig. 3. Percent of time inside or outside the stimulus area in the recall test that was spent in nipple-search behavior by pups with whiskers (W+) and by pups without whiskers (W). The inset shows the ellipse (20% of the arena surface) enclosing the three spots of Chanel that was used to define the stimulus area. Means and SEs are given.
whiskers disrupts a variety of behaviors (Brecht et al. 1997; Cardenas et al. 2001; Krupa et al. 2001). The steps leading to successful suckling are among the most complex and precise orienting behaviors performed by newborn rabbits, requiring them to rapidly establish and maintain contact with the mother’s ventrum, to perform a particular pattern of head movements through her fur, to locate a nipple, to grasp and remain attached to it, and this repeatedly since the pups typically switch nipples several times within one nursing episode (Hudson and Distel 1983). As for the altricial young of other mammals (Hofer et al. 1981; Larson and Stein 1984; Blass et al. 1988; Morrow–Tesch and McGlone 1990) somatosensory input from the perioral area is critical for successful suckling. However, in rabbit pups this seems to come from the skin and lips rather than from the whiskers, and also seems to apply to the reinforcing role of perioral stimulation in learning to associate a novel odor with suckling (Hudson et al. 2002). Although the results are consistent with our failure to find evidence of movement of the
whiskers or of ‘‘whisking’’ in these young animals, they are surprising given the finding that rat pups rapidly learn to associate whisker stimulation with a novel reinforcer (Landers and Sullivan 1999a, b), and after cutting the whiskers on postnatal day 3 or 4, take longer to attach to nipples than intact controls (Young and Sullivan, congress abstract, cited in Landers and Sullivan 1999a). The lack of effect in rabbit pups may be due to their extreme dependence on odor cues on the mother’s ventrum for the release and guidance of the stereotyped nipple-search behavior (Hudson and Distel 1983, 1995; Distel and Hudson 1985).
Acknowledgements Support from PAPIIT Grant IN217100 is gratefully acknowledged and we thank Daniel Paul Alarcon Tinoco for photographic assistance in preparing Fig. 1, and Hans Distel for help with the German abstract.
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Zusammenfassung Tragen die Schnurrhaare bei neugeborenen Kaninchen (Oryctolgus cuniculus) zum Zitzensuchund Saugverhalten bei? Jungkaninchen werden nur einmal am Tag fu¨r etwa drei Minuten gesa¨ugt. Fu¨r das Auffinden der Zitzen sind sie auf ein Pheromon auf der Bauchhaut der Mutter angewiesen, und fu¨r deren Ergreifen auf periorale taktile Reize. In Fortsetzung unserer Untersuchungen zur sensorischen Kontrolle dieses Verhaltens beschreiben wir die Anordnung der Schnurrhaare bei neugeborenen Kaninchen und untersuchen ihren mo¨glichen Beitrag zum Sa¨ugeverhalten. Bei der Geburt besitzen Jungkaninchen etwa 76, in 7–9 Reihen angeordnete Schnurrhaare, deren La¨nge von rostral nach caudal zunimmt. Nach Abschneiden der Schnurrhaare wurden keine signifikanten Unterschiede zwischen Jungen mit und ohne Schnurrhaare beobachtet, sowohl in der Latenz des Zitzensuchens, des Zitzenfassens, wie in der Besaugungsdauer, der erhaltenen Milchmenge oder dem Grad der Konditionierung auf einen neuen Geruchsstoff. Demnach spielen bei neugeborenen Kaninchen die Schnurrhaare keine wesentliche Rolle fu¨r das Auffinden und Besaugen von Zitzen. r 2005 Elsevier GmbH. All rights reserved.
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Authors’ address: Adela Mendoza, Valeria Guzma´n, Carolina Rojas, and Robyn Hudson, Departamento de Biologı´ a Celular y Fisiologı´ a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Apartado Postal 70228, CP 04510 Me´xico, D.F., Mexico. (e-mail: [email protected].)